Tankyrase (TNKS) is a member of the poly-ADP-ribose polymerase (PARP) family, which uses NAD+ as a substrate to transfer ADP-ribose polymers onto target proteins, resulting in a post-translational modification referred to as PARsylation. TNKS was first identified as a binding partner for telomerase repeat binding factor 1 (TRF1), which is a key player in the regulation of telomere length at the chromosome ends. Telomere length is maintained by the reverse transcriptase telomerase. The TRF1 and TRF2 proteins are DNA binding proteins that regulate the length and stability of telomeres. Poly-ADP-ribosylation (PARsylation) of TRF1 by TNKS inhibits the ability of TRF1 to bind telomeric DNA thereby allowing telomerase access to telomeric DNA. Thus, TNKS proteins function as positive regulators of telomere length. In addition, it has been reported that TNKS regulates sister chromatid separation during mitosis as well as vesicle trafficking. Additional binding partners of TNKS have recently been identified including CASC3 and BLZF1 (Golgin-45) suggesting roles for TNKS in diverse cellular processes including mRNA metabolism and Golgi structure maintenance.
There are two TNKS genes, TNKS1 and TNKS2, in the human and mouse genomes. Individual and double-knockout of TNKS1 and TNKS2 in mice suggests that they share significant functional redundancy (Chiang Y. J. et. al., “Tankyrase 1 and Tankyrase 2 are Essential but Redundant for Mouse Embryonic Development,” PLoS ONE 3(7): e2639. Pp. 1-10 (2008)) as the single homozygous TNKS1 or TNKS2 mice had relatively mild growth phenotypes and no defects in telomere maintenance, whereas the double knockout caused early embryonic lethality.
More recently, TNKS proteins were shown to bind directly to AXIN1 and AXIN2 proteins, which are negative regulators of the Wnt pathway, and regulate their steady state levels by PARsylation and ubiquitination (Huang, S. M. et al. “Tankyrase Inhibition Stabilizes Axin and Antagonizes Wnt Signalling,” Nature, 461, pp 614-620 (2009)). Small-molecule inhibitors of tankyrases TNKS1 and TNKS2 can downregulate Wnt signaling (Huang, S. M. et al. “Tankyrase Inhibition Stabilizes Axin and Antagonizes Wnt Signalling,” Nature, 461, pp 614-620 (2009); Chen, B. et. al. “Small Molecule-Mediated Disruption of Wnt-Dependent Signaling in Tissue Regeneration and Cancer,” Nature Chem. Biol., 5(2), pp 100-107, (2009)) in several immortalized and malignant human cell lines. These inhibitors of TNKS also regulate the Wnt pathway in vivo in a Wnt signaling-dependent zebrafish model of fin regeneration.
Signaling by the Wnt family of secreted proteins plays an essential, evolutionarily conserved role in embryonic development and adult tissue homeostasis in a vast array of organisms including, flies, worms, chickens, frogs and mammals. Wnt signaling is a fundamental morphogenetic pathway that is deployed in diverse settings throughout development to regulate processes such as cell fate specification, tissue patterning, polarity, gastrulation, stem cell maintainence, and cell migration. The Wnt pathway is extraordinarily complex and the binding of Wnt ligands can lead to a variety of biological outcomes depending on the molecular and cellular context. Wnt signaling is often described in terms of either the canonical pathway or one of several non-canonical pathways.
In the canonical or β-catenin-dependent Wnt pathway, specific Wnt ligands regulate the level and sub-cellular localization of β-catenin. In the absence of an activating Wnt signal, glycogen synthase kinase 3β (GSK3β) collaborates with the AXIN and APC (adenomatous polyposis coli) proteins and other factors to phosphorylate β-catenin at its amino (N)-terminal domain. The phosphorylated β-catenin is recognized and ubiquitinated by a complex containing a β-transducin repeat-containing protein (βTrCP), and is then degraded by the proteasome. Wnt binding to the Frizzled-low density lipoprotein-related protein (LRP)-5/6 co-receptor complex on the cell surface leads to the recruitment of disheveled and the inhibition the AXIN/GSK3β complex. This, in turn, leads to the stabilization of the free pools of β-catenin which can enter the nucleus, bind to T cell factor (TCF) transcriptional regulators along with other cofactors and modulate transcription of various genes.
Wnt pathway deregulation has been implicated in many human diseases including cancer as well as many non-oncogenic disorders such as cardiac disease, osteroporosis, osteoarthritis, diabetes, fibrotic/proliferative diseases, Alzheimer's disease and schizophrenia. Wnt/β-catenin signaling is of particular relevance to colorectal cancers (CRC), which are the second leading cause of cancer death in Western societies. Mutations in the tumor suppressor gene, APC, are responsible for Familial Adenomatous Polyposis. Truncating mutations APC are also the most prevalent genetic alterations in sporadic CRC. Inactivating mutations of AXIN1/2 and oncogenic mutations in β-catenin, all of which lead to the stabilization of β-catenin and to altered expression of β-catenin/TCF-regulated genes in the absence of exogenous Wnt signals, have also been identified in human cancers including CRC. Indeed, aberrant activation of Wnt/β-catenin signaling is likely an obligatory step in the initiation of the majority, if not all, human CRC.
There is also good evidence for Wnt pathway hyperactivation in the initiation and/or progression on a variety of other human cancers including gastric, pancreatic, kidney (Wilms), medulloblastoma, melanoma, lung, thyroid, breast and prostate cancer. This pathway activation is achieved by either oncogenic mutations in β-catenin, or loss of function mutations in APC or AXIN. In addition, mutations or epigentic silencing of extracellular negative regulators such as SFRP's, DKK's and WIF can also lead to abnormal pathway activity and have been widely reported in a large number of different human cancers.
Blocking canonical Wnt signaling in CRC and other Wnt-dependent tumors such as lung, breast and teratocarcinomas has been shown to inhibit tumor growth in human xenografts grown in mice or in transgenic mouse models. Several classes of small molecules have been shown to act as Wnt signaling inhibitors at various “nodes” of the pathway including the disruption of dishevelled activity and b-catenin interaction surfaces with TCF/LEF or pygopus. The efficient assembly of the multi-protein β-catenin destruction complex is dependent on the steady-state levels of its principal constituents. AXIN has been reported to be the concentration-limiting factor in regulating the efficiency of the β-catenin destruction complex and overexpression of AXIN induces β-catenin degradation even in cell lines expressing truncated APC (Hart, M. J. et. al. “Downregulation of Beta-Catenin by Human Axin and its Association with the APC Tumor Suppressor, Beta-Catenin and GSK3 Beta,” Curr. Biol., 8(10) pp 573-581 (1998)). Thus, AXIN protein levels need to be tightly regulated to ensure proper Wnt pathway signaling and regulation of AXIN stability by TNKS therefore represents a good therapeutic target.
Cancer stem cells, found in many types of cancer, are rare populations of malignant cells with the capacity for endless self-renewal. They are believed to be responsible for tumor growth, recurrence and metastasis. Also referred to as “tumor-initiating cells,” these cells have been identified in many types of solid tumor cancers, including cancer of head and neck, breast, lung, prostate, pancreas and glioblastoma. Cancer stem cells appear to be preferentially resistant to both standard chemotherapy and radiotherapy. One important therapeutic strategy is to specifically target the key biological pathways which are thought to be critical to the activity and survival of cancer stem cells. Since the Wnt pathway has been shown to be critical for cancer stem cells in many types of malignancies (e.g. squamous cancer stem cells), TNKS inhibitors are promising therapeutic compounds for use in treating human disease where cancer stem cells are thought to play a role (i.e. in recurrent or resistant disease). TNKS inhibitors could be either used alone or in combination with current chemotherapies.
Upregulation of telomerase and telomere maintenance is necessary for most cancer cells to replicate indefinitely and thereby enable tumor growth and metastasis. One strategy for the development of anti-cancer therapies is to inhibit telomerase activity in cancer cells. Inhibiting telomerase activity should result in telomere shortening which can cause senescence and death of cancer cells. Another, strategy to inhibit the telomere elongation in cancer cells would be to effectively inhibit telomerase by exclusion by preventing the PARsylation of TRF1 by TNKS. Thus, TNKS inhibitors would be suitable cancer therapies either alone or in combination with telomerase inhibitors by targeting teleomeres and driving cancer cells towards senescence.
A need therefore exists for potent and selective inhibitors of TNKS that may be used to treat cancer. The present application discloses such potent and selective tankyrase inhibitors with good drug-like properties that are suitable for inhibiting the growth of cancer cells. These compounds are especially appropriate for inhibiting CRC or any other human tumor that has evidence of Wnt pathway activation and/or dependence.